Conditions have been established which allow the isolation of rat adipose cell plasma membranes retaining a large part of the stimulatory effect of insulin in intact cells. Studies with these membranes suggest that in addition to stimulating the translocation of glucose transporters to the plasma membrane, insulin appears to induce a structural or conformational change in the glucose transporter manifested in an altered activation energy for plasma membrane glucose transport and possibly in an altered immunoreactivity as assessed by Western blotting. To examine the possible role of protein kinase C in the signaling mechanism of insulin-stimulated glucose transport in the isolated rat adipose cell, we have compared the effects of insulin and the tumor promoting phorbol ester, phorbol myristate acetate (PMA), on 3-0- methylglucose transport activity and on the distribution of D- glucose-inhibitable cytochalasin B binding sites in rat adipose cells. The data suggest that 1) protein kinase C activation causes the translocation of glucose transporters from an intracellular, low-density microsomal pool to the plasma membrane without a corresponding increase in transport activity and 2) insulin appears to cause the activating of these translocated glucose transporter proteins. We have addressed the question of a long term effect of insulin on adipose cell glucose transporter content by using a cultured cell system, the 3T3-F442A preadipocyte. At confluence, cells were made to differentiate without or with insulin for 15 days. This study, besides documenting the acute effect of insulin on glucose transporter translocation in 3T3-F442A adipose cells, clearly demonstrates that chronic exposure to insulin of these differentiating cells markedly increases the content of glucose transporters. A procedure for purification of the 45 kDa transport protein from rat brain has been developed. An approximately 5,000- fold purification of the rat brain glucose transporter has been achieved with a yield of 25%.